The present invention relates to novel short interfering RNA (siRNA) molecules that are multi-targeted. More specifically, the present invention relates to siRNA molecules that are capable target two or more sequences. In one embodiment, multi-targeting siRNA molecules are designed to incorporate features of siRNA molecules and features of micro-RNA (miRNA) molecules. In another embodiment, multi-targeting siRNA molecules are designed so that each strand is directed to separate targets.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
RNA interference (RNAi) is a process where double-stranded RNA triggers together with protein complexes downregulate mRNA complementary to the triggers (Hannon and Rossi, 2004). The triggers are 19 nt long RNA duplexes with 2 nt 3′ overhangs and are referred to as short interfering RNAs (siRNAs). The protein complex, known as the RNA-induced silencing complex (RISC), incorporates one of the siRNA strands and RISC uses this strand as a template to recognize target mRNA. RISC then cleaves mRNA with perfect or near-perfect complementarity to the guide strand. Short interfering RNAs are used as tools to down-regulate specific genes and can either give transient or—when stably integrated as short hairpins RNAs (shRNAs) (Paddison et al., 2002)—stable suppression.
The siRNAs and shRNAs resemble intermediates in the processing pathway of the endogenous microRNA (miRNA) genes (Bartel, 2004). Indeed, siRNAs can function as miRNAs and vice versa (Zeng et al., 2002; Doench et al., 2003). MicroRNAs, like siRNAs, use RISC to downregulate target genes, but unlike siRNAs, most animal miRNAs do not cleave the mRNA. Instead, miRNAs reduce protein output through translational suppression or polyA removal and mRNA degradation (Wu et al., 2006). Known miRNA binding sites are within mRNA 3′ UTRs; miRNAs seem to target sites with near-perfect complementarity to nucleotides 2-8 from the miRNA's 5′ end (Rajewsky, 2006; Lim et al., 2005). This region is known as the seed region. Because siRNAs and miRNAs are interchangeable, exogenous siRNAs will downregulate mRNAs with seed complementarity to the siRNA (Birmingham et al., 2006. Multiple target sites within a 3′ UTR give stronger downregulation (Doench et al., 2003).
Although many siRNA molecules have been developed for the treatment of cancer disease, and other conditions, there remains a need for the development of additional new molecules and methods for the RNAi treatment of cancer, disease, and other conditions, particularly the development of new siRNA molecules.